Bottom Line:
The release of MPs was highly regulated and dependent on caspase 3 and Rho-associated kinase.Further analysis identified these MPs as a central chemoattractant in vitro and in vivo.In addition, intravenously transplanting circulating MPs from the ob/ob mice lead to activation of monocytes in circulation and adipose tissue of the wild type mice.

Affiliation: Department of Pediatrics, University of California San Diego (UCSD), La Jolla, California, United States of America.

ABSTRACTMacrophage infiltration of adipose tissue during weight gain is a central event leading to the metabolic complications of obesity. However, what are the mechanisms attracting professional phagocytes to obese adipose tissue remains poorly understood. Here, we demonstrate that adipocyte-derived microparticles (MPs) are critical "find-me" signals for recruitment of monocytes and macrophages. Supernatants from stressed adipocytes stimulated the attraction of monocyte cells and primary macrophages. The activation of caspase 3 was required for release of these signals. Adipocytes exposed to saturated fatty acids showed marked release of MPs into the supernatant while common genetic mouse models of obesity demonstrate high levels of circulating adipocyte-derived MPs. The release of MPs was highly regulated and dependent on caspase 3 and Rho-associated kinase. Further analysis identified these MPs as a central chemoattractant in vitro and in vivo. In addition, intravenously transplanting circulating MPs from the ob/ob mice lead to activation of monocytes in circulation and adipose tissue of the wild type mice. These data identify adipocyte-derived MPs as novel "find me" signals that contributes to macrophage infiltration associated with obesity.

pone.0123110.g001: Attraction of monocytes and macrophages to supernatants of mature adipocytes exposed to palmitic acid.Migration of (A) RAW264.7 cells or (B) primary mouse macrophages through a transwell (8 μm pore size) to supernatants from untreated (control) or treated differentiated mature adipocytes with 0.5mM palmitic acid. MCP-1 (50 ng/ml) was used as positive control. (C) Adipocytes were treated with 0.5 mM palmitic acid in the absence or presence of 0.025 U/ml apyrase, 0.5 U/ml phospholipase-D or 50 μg/ml control IgG or MCP-1 neutralizing antibody. Macrophages that migrated to the lower chamber were stained with DAPI and the number of cells was counted under fluorescence microscopy. Values represent as mean ± S.D. of representative experiment. * P < 0.05; ** P < 0.01; ***P < 0.001 compared to controls.

Mentions:
It has long been speculated that in higher organisms where dying cells and phagocytes are not located in immediate proximity, dying cells secrete compounds that recruit monocyte and macrophages to the site of death. To determine whether stressed or dying adipocytes release such factors, we initially assessed cell-free supernatants of mature adipocytes exposed to the lipotoxic free fatty acids, palmitic acid, that are typically present in the obese adipose tissue for their ability to attract RAW 264.7 cells or primary mouse macrophages in transwell migration assays (Fig 1A and 1B). Supernatants from adipocytes treated with palmitic acid resulted in marked increase in monocyte migration which was more than 3-fold higher than controls and greater than the migration elicit by the potent chemotactic factor MCP-1 (Fig 1A and 1B). In order to determine the nature of the attraction signals release by adipocytes, we next access two previously described “find me” signals in other model systems including extracellular nucleotides, and the phospholipid LPC, as well as MCP-1, which has been proposed to contribute to macrophage infiltration into obese adipose tissue in vivo as potential candidates [29]. Treatment of supernatants from adipocytes with either apyrase or phospholipase D, enzymes that hydrolyse nucleotides or LPC respectively, as well as neutralizing anti-MCP-1 antibodies did not affect the chemotactic activity of palmitic acid treated adipocytes (Fig 1C).

pone.0123110.g001: Attraction of monocytes and macrophages to supernatants of mature adipocytes exposed to palmitic acid.Migration of (A) RAW264.7 cells or (B) primary mouse macrophages through a transwell (8 μm pore size) to supernatants from untreated (control) or treated differentiated mature adipocytes with 0.5mM palmitic acid. MCP-1 (50 ng/ml) was used as positive control. (C) Adipocytes were treated with 0.5 mM palmitic acid in the absence or presence of 0.025 U/ml apyrase, 0.5 U/ml phospholipase-D or 50 μg/ml control IgG or MCP-1 neutralizing antibody. Macrophages that migrated to the lower chamber were stained with DAPI and the number of cells was counted under fluorescence microscopy. Values represent as mean ± S.D. of representative experiment. * P < 0.05; ** P < 0.01; ***P < 0.001 compared to controls.

Mentions:
It has long been speculated that in higher organisms where dying cells and phagocytes are not located in immediate proximity, dying cells secrete compounds that recruit monocyte and macrophages to the site of death. To determine whether stressed or dying adipocytes release such factors, we initially assessed cell-free supernatants of mature adipocytes exposed to the lipotoxic free fatty acids, palmitic acid, that are typically present in the obese adipose tissue for their ability to attract RAW 264.7 cells or primary mouse macrophages in transwell migration assays (Fig 1A and 1B). Supernatants from adipocytes treated with palmitic acid resulted in marked increase in monocyte migration which was more than 3-fold higher than controls and greater than the migration elicit by the potent chemotactic factor MCP-1 (Fig 1A and 1B). In order to determine the nature of the attraction signals release by adipocytes, we next access two previously described “find me” signals in other model systems including extracellular nucleotides, and the phospholipid LPC, as well as MCP-1, which has been proposed to contribute to macrophage infiltration into obese adipose tissue in vivo as potential candidates [29]. Treatment of supernatants from adipocytes with either apyrase or phospholipase D, enzymes that hydrolyse nucleotides or LPC respectively, as well as neutralizing anti-MCP-1 antibodies did not affect the chemotactic activity of palmitic acid treated adipocytes (Fig 1C).

Bottom Line:
The release of MPs was highly regulated and dependent on caspase 3 and Rho-associated kinase.Further analysis identified these MPs as a central chemoattractant in vitro and in vivo.In addition, intravenously transplanting circulating MPs from the ob/ob mice lead to activation of monocytes in circulation and adipose tissue of the wild type mice.

Affiliation:
Department of Pediatrics, University of California San Diego (UCSD), La Jolla, California, United States of America.

ABSTRACTMacrophage infiltration of adipose tissue during weight gain is a central event leading to the metabolic complications of obesity. However, what are the mechanisms attracting professional phagocytes to obese adipose tissue remains poorly understood. Here, we demonstrate that adipocyte-derived microparticles (MPs) are critical "find-me" signals for recruitment of monocytes and macrophages. Supernatants from stressed adipocytes stimulated the attraction of monocyte cells and primary macrophages. The activation of caspase 3 was required for release of these signals. Adipocytes exposed to saturated fatty acids showed marked release of MPs into the supernatant while common genetic mouse models of obesity demonstrate high levels of circulating adipocyte-derived MPs. The release of MPs was highly regulated and dependent on caspase 3 and Rho-associated kinase. Further analysis identified these MPs as a central chemoattractant in vitro and in vivo. In addition, intravenously transplanting circulating MPs from the ob/ob mice lead to activation of monocytes in circulation and adipose tissue of the wild type mice. These data identify adipocyte-derived MPs as novel "find me" signals that contributes to macrophage infiltration associated with obesity.